Central issues in vertebrate embryonic heart formation include patterning along the anterior-posterior (A/P) axis, formation of heart-chamber specific cardiomyocytes, and the acquisition of specific cell functions, i.e., regulation of cell differentiation. This proposal addresses these issues focusing on the formation of distinct cardiomyocyte types prior to and during heart tube formation, and as heart chambers form, emphasizing atrial specification. A major aim is to delineate the molecular mechanisms involved in atrial cardiomyocyte formation. Mechanisms that regulate atrial gene expression are much less well understood than those for the ventricle. Our observations support the hypothesis that cardiomyocytes initially express an atrial-like phenotype that is suppressed during chamber formation of the ventricles. Thus the atrial phenotype is the default pathway for the heart. These observations are based on our isolation and characterization of an atrial-specific slow myosin heavy chain gene, slow MyHC 3 (SM3), a homolog of the chicken AMHC1. Initially, the SM3 gene is expressed throughout the tubular heart, but as the heart tube loops and chamberizes, expression disappears from the ventricles, remaining in the atrium. We have shown that 840 bp of the SM3 promoter upstream from the transcription start site, is sufficient for correct spatial and temporal atrial-specific expression in cardiomyocytes, in the avian heart in ovo, and in hearts of transgenic embryonic and adult mice. Within this 840 bp is a 160 bp enhancer, designated Atrial Regulatory Domain 1 (ARD1), sufficient to drive atrial-specific reporter expression in ovo. Deletion and mutation within the ARD1, coupled with transient transfection of atrial and ventricular cardiomyocytes, identified three elements responsible for atrial specificity: a vitamin D receptor binding site (VDRE), an overlapping retinoic acid receptor binding site (RARE), and a GATA factor binding site. Whereas the GATA element is an essential positive cardiac regulator, the VDRE/RARE functions specifically in the ventricles to suppress expression. Control of these elements is complex. We find that the product of the Iroquois homeobox gene, IRX4 acts within the ARD1 to suppress expression of the SM3 gene, as does the vitamin D receptor (VDR/RXR); whereas the calcineurin and protein kinase C alpha (PKCalpha) pathways activate expression of the SM3 gene. We find that VDR/RXR and IRX4 are essential for specification of cardiac precursors to a ventricular fate instead of an atrial fate, thus the interaction of the nuclear hormone receptors with IRX4, and with the calcineurin and PKCalpha pathways is central to specification of the atrial phenotype.